Introduction


The receptors TSHr, FSHr and LHr or hCGr are members of the rhodopsin-like G protein-coupled receptor (GPCR) family.
FSH is secreted from the pituary gland to regulate reproduction in mammals and acts on the FSH receptor. In females, it induces maturation of ovarian follicles and in males it stimulates sertoli cell proliferation in testes and supports spermatogenesis.
LHr/hCGr is expressed on gonadal cells and is required for normal reproductive function in males and females and for male sexual differentiation.
Growth and function of the thyroid are regulated by thyrotropin (TSH) secreted by the pituary gland and acts on the thyrotropin receptor (TSHr).
In contrast to LHr/hCGr and FSHr, the TSHr has been shown to present significant constitutive activity (1,2).

As members of the GPCR superfamily, they contain a serpentine region (7TM) containing seven transmembrane helices with many of the sequences signatures typical of this receptor family (Figure 1 and 2).

Figure 1: Cartoon representation of the domain structure of the Glycoprotein Hormone Receptors.

In addition, they contain a large (350-400 residues) N-terminal ectodomain that is responsible for the high affinity and selective binding of the corresponding hormones (TSH, FSH and LH or hCG, respectively)(Figure 1 and 2)(3,4,5).
Typical for the glycoprotein hormone receptors is that they harbour an extremely conserved SHCCAF motif implicated in activation (6) and a YDY sulfation motif (7) in between the ectodomain and the serpentine portion (Figure 1)

Figure 2: The FSHr ectodomain (purple) in complex with the FSH hormone (red and green) (PDB 1XWD) and below in a random position a homology model of the FSHr serpentine domain (blue).

After specific hormone binding to the ectodomain, the serpentine region is capable of activating a G protein (8).
The intramolecular transduction of the signal between these two regions of the receptors is currently a heavily studied topic.

Structure of GpHRs hormone in more detail


The hormones (TSH,FSH and LH/hCG) are heterodimeric cysteine-knot folded proteins, which all share a common alpha subunit, but differ from each other through the beta subunit. The subunits are not covalently linked, but cluther together through rings of disulfide bonds (9,10).

Figure 3: hCG hormone (PDB 1HCN) indicating the alpha chain in green and the beta chain in red. Disulfide rings are colored as yellow sticks.

Structure of GpHRs ectodomain in more detail


The 3D structure of the ectodomain of the FSHr in complex with the FSH hormone was recently solved (Figure 4) (11) and models of the TSHr in complex with the TSH and LHr/hCGr in complex with LH, which can be found in this database system were built using the FSHr/FSH complex.

Figure 4: The FSHr ectodomain (purple) in complex with the FSH hormone (red and green) (PDB 1XWD) indicating the alpha chain in green and the beta chain in red.

The ectodomain has a leucine-rich repeat structure (LRR). LRRs are 20-25 residue protein motifs consisting of a beta strand and an alpha helix connected by a turn. When assembled sequentially in a protein, the LRRs determine a horseshoe-like structure with the beta strands making a concave inner surface. This surface has been shown in several publications to harbour the binding interface with the hormone and this has been confirmed with the crystallization of the FSHr/FSH complex (11). Strikingly, the FSHr ectodomain does not display a true strand-turn-helix LRR structure, but instead strand-turn-coil, where the helical segment is degenerated to a coily structure (Figure 5).

The structure of the beta strand of such a leucine-rich repeat consists of two mostly hydrophobic residues pointing to the core (residue numbers 3049 and 3051 where "30" stands for the third repeat) and preferably polar residues pointing outwards available for interaction with the hormone (numbers 3047, 3048, 3050, 3052 and 3053)(Figure 5).

Figure 5: Structure of the third leucine-rich repeat of the FSHr (PDB 1XWD) indicating the coiled outer surface and the concave inner side consisting of a beta strand. Residues are numbered using the polar residue in between the two hydrophobics as a reference point "3050" (see the numbering section for mode details).

Flanking the LRR region of the ectodomain are two cysteine-rich domains, of which the 3D structure of the upstream cluster can be seen in the FSHr/FSH complex (11), but the structure of the downstream cluster remains completely unknown.
Since the cysteine cluster cannot be reliably aligned throughout all GpHRs, it is not included in the models here available.

Structure of GpHRs serpentine region in more detail


As they belong to the rhodopsin-like GPCR family and display many of the signatures in primary structure, the serpentine regions of GpHRs are modelled from the crystal structure of bovine rhodopsin, the only GPCR structure available today (Figure 6) (12,13).
This domain consists of seven helices perpendicular to the plasma membrane intervened by intra- and extracellular loops. An shorter eight helix lies parallel to the membrane on the cytoplasmic side right after transmembrane helix 7 (Figure 6).

Figure 6: The crystal structure of bovine rhodopsin on the left (PDB 1GZM) including retinal (magenta) and the inner and outer loops. On the right, a homology model of the FSHr serpentine domain is displayed. Only the most conserved parts for which a reliable alignment is available can be modeled. As the picture shows, these parts are the seven transmembrane helices and the last eighth helix. Inner and outer loop modeling is unreliable and these parts cannot be modeled.

The primary transmembrane helix signatures are displayed in Figure 1. These are almost the same as for rhodopsin, except in helix 5, where the conserved proline is substituted by an alanine in GpHRs.

Activation mechanism of the serpentine region of GpHRs


Since GpHRs share lots of common primary structure features with rhodopsin, they are also likely to share common mechanisms of activation with rhodopsin. Crystallographic data is only available for the inactive conformation of rhodopsin (12,13), but nevertheless molecular activation mechanisms that are based on a panel of experimental approaches involving site-directed mutagenesis, cross-linking and molecular modeling have been proposed (14).
In addition, over the past ten years, LHr/CGr and even more TSHr has been found to be activated by a wide spectrum of gain-of-function mutations, leading to pathogenic conditions in many cases (15).
Molecular modeling studies predict that these activating mutations are likely to loosen certain interhelical interactions and hereby breaking one or more bonds, instead of creating novel interactions by the mutated residue (16,17).

Dimerization


Bovine rhodopsin was crystallized as a homodimer, albeit a non-physiological one, since both protomers are upside down compared to each other (see the location of Helix 8 in Figure 7).

Figure 7: The dimer of the bovine rhodopsin crystal (PDB 1GZM). Both protomers are upside down as can be seen on the location of the cytoplasmic Helix 8, which is on opposite sides.

However, work on Class 3 GPCRs (18) has proven dimerization as an obligatory phenomenon. The discovery that even Class 1 GPCRs are also capable of homo- and heterodimerization (19) has added additional complexity. Recently, it was proven that the TSHr forms homodimers in the plasma membrane of living cells and that the dimeric interactions are likely to occur through the heptahelical portion of the receptor (20). Even functional complementarity, restoring TSH responsiveness of two different loss-of-function mutants, when coexpressed in the same cell, implied that a TSHr dimer has the capability to work as a single functional unit (20). Similar results have been reported for the LHr/CGr and FSHr (21,22,23). In addition to homodimerization, experiments demonstrate that it is possible to generate TSHR-LH/CGr heterodimers.

Allosterism


A second conclusion from Urizar et al. (20) is that hormone binding to their receptors displays negative cooperativity. In short, this means that radioactive I125-TSH can be desorped from a TSHr-LHr/CGr heterodimer by adding an excess of hCG. An allosteric mechanism is suggested to be at play here, because in such a heterodimer, each hormone can only bind to the ECD of its own receptor. This supports the hypothesis that hormone binding on one protomer could induce a conformational change in the other protomer, hereby losing its affinity for its own hormone.

Predict the effect of a GpHR mutation on-line!


Mutation studies combined with molecular modeling is thus an invaluable tool to study the activation mechanism of the Glycoprotein Hormone Receptors. This is the aim of the Glycoprotein Hormone Receptors Information System (24). It combines mutagenesis data with 3D structural information to study the putative effect of a mutation in silico and on-line.

References


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All molecular images on this page were created using the YASARA program.